[Cardiac arrest during the concomitant administration of sulprostone and noradrenaline].
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Biomedical subjects
Publications and source records attributed to G Fabre.
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1. The aims were to refine experimental conditions (using 76 human hepatocyte preparations) in terms of the selection of enzyme inducers and their optimal concentration, the treatment duration with inducers and the choice of specific cytochrome P450 isoform(s) probes to optimize the use of primary hepatocytes for predicting the potential induction by new chemical entities of cytochrome P450 isoforms in vivo in man. 2. In the absence of any inducer, basal cytochrome P450 isoform(s)-mediated activities decreased to 20% of their initial activity (end of the seeding period) by 72-96 h. In contrast, UGT-dependent enzyme activities remained at a constant level (+/- 20%) up to the fifth day of culture. 3. Beta-naphthoflavone, at an optimal concentration of 50 microM and after a 3-day treatment, specifically and potently induced 7-ethoxyresorufin (10.4 +/- 10.4-fold, n = 74) and phenacetin (6.6 +/- 6.4-fold, n = 60) O-deethylation processes, markers for CYP1A1 and CYP1A2 isoforms respectively. Only a 2-fold increase was noted following treatment with 2 mM phenobarbitone, whereas dexamethasone and rifampicin had no effect at all. 4. A 3-day treatment of human hepatocytes with 50 microM dexamethasone was associated with a major induction of both coumarin 7-hydroxylation (9.4 +/- 11.4-fold, n = 49) and nifedipine dehydrogenation (4.7 +/- 3.8-fold, n = 61), markers for CYP2A6 and CYP3A4 respectively. Phenobarbitone, however, exhibited a broad but moderate inducing effect on 7-ethoxyresorufin (2.2 +/- 1.5-fold, n = 55) and phenacetin (1.7 +/- 0.9-fold, n = 54) O-deethylation, coumarin 7-hydroxylation (3.9 +/- 9.2-fold, n = 50) and nifedipine dehydrogenation (2.1 +/- 2.0-fold, n = 47). 5. Km obtained for the different cytochrome P450 isoform substrates in untreated hepatocytes were in the same range of magnitude that those determined on human hepatic microsomal fractions. Enzyme induction processes were characterized by a large increase in apparent Vmax whereas apparent Km were not affected. 6. These studies demonstrate that human hepatocytes in primary culture can respond specifically and quantitatively to model inducers. This in vitro system offers a useful approach to study the regulation of human hepatic biotransformation activities and should facilitate the demand for a reproducible method for addressing cytochrome P450 induction.
OBJECTIVE: An anti-hepatitis A virus seroprevalence survey was performed in 1997 in 1052 French army recruits (mean age: 21.2 years). To describe epidemiological trends, the current pattern was compared to previous results obtained by similar methods in 1985, 1990 and 1993. RESULTS: In 1997, overall anti-hepatitis A virus seroprevalence was 11.5%. The greatest risk factor of hepatitis A infection was related to travel in intermediate or highly endemic areas for hepatitis A virus: 46% of overseas residents (odds ratio = 10.3), 28% of recruits who had travelled in developing countries (odds ratio = 3.7) and 7.65% of French living in industrialised countries are anti-hepatitis A virus antibody positive. Moreover, seroprevalence was higher in subjects with a history of icteria (adjusted odds ratio = 3.5) and families with at least 3 children (adjusted odds ratio = 3). No association was found with drinking water, socioeconomic status such as baccalaureat degree, or parents profession. The seroepidemiological shift of hepatitis A, as assessed in three previous studies, shows a marked decrease of 20% in 12 years from 30.4% in 1985, to 21.3% in 1990, to 16.3% in 1993, and to 11.5% in 1997. The decrease in the prevalence of anti-hepatitis A virus was more marked in young adults who had never travelled in endemic countries (decrease of 20%) than those who had visited or lived in developing countries (decrease of 10%). CONCLUSION: Although France is not highly endemic for hepatitis A thanks to improved hygiene and housing conditions over the past 20 years, a pattern of intermediate endemicity was seen in French overseas areas in which the risk of outbreaks of hepatitis A was higher. The decrease in anti-hepatitis A virus seroprevalence in French youth can be used to draft a public health policy for hepatitis A control.
The oxidative metabolism of irbesartan, a new nonpeptide angiotensin II receptor antagonist, was investigated on 12 human fully characterized hepatic microsomes and purified cytochrome P-450 (CYP) isoforms. After incubation of microsomes with irbesartan and NADPH, four main hydroxy metabolites were formed, as confirmed by liquid chromatography-mass spectrometry analysis. Irbesartan oxidation follows Michaelis-Menten kinetics, consistent with the involvement of a single CYP isoform in these hydroxylation processes. Only a low interindividual variability (2-fold difference) was observed in drug oxidation, even in preparations lacking CYP2D6. Km and Vmax for irbesartan oxidation were 54 +/- 6.5 microM and 0.62 +/- 0.18 nmol/min/mg, respectively. Irbesartan oxidation correlated (r2 = 0. 769) with tolbutamide (CYP2C9 substrate) 4-methyl-hydroxylation. Oxidation of irbesartan was markedly inhibited by sulfaphenazole (CYP2C9 inhibitor), but not by any of several other CYP inhibitors. In the same manner, both tolbutamide and warfarin (CYP2C9 substrates), were competitive-type inhibitors of irbesartan oxidation with Ki values of 500 and 30 microM, respectively. Moreover, irbesartan was a competitive-type inhibitor of tolbutamide 4-methylhydroxylation (Ki = 317 microM). Nifedipine also potentially decreased irbesartan oxidation, whereas neither ketoconazole and triacetyloleandomycin (CYP3A inhibitors), nor diltiazem and verapamil, (CYP3A4 substrates), exhibited an inhibitory effect. Additional studies demonstrated that nifedipine was an inhibitor of irbesartan (Ki = 20 microM) and tolbutamide oxidation processes, whereas irbesartan had no effect at all on nifedipine dehydrogenation. Enzyme kinetics suggest that nifedipine is a noncompetitive-type inhibitor of CYP2C9-mediated catalytic activities. Finally, only microsomes containing recombinant human liver CYP2C9 were capable of oxidizing irbesartan. These results provide evidence that CYP2C9 plays a major role in irbesartan oxidation.
PURPOSE: To determine and compare the relationship between in vivo oral absorption in humans and the apparent permeability coefficients (Papp) obtained in vitro on two human intestinal epithelial cell lines, the parental Caco-2 and the TC-7 clone. METHODS: Both cell lines were grown for 5-35 days on tissue culture-treated inserts. Cell monolayers were analysed for their morphology by transmission electron micrography, and for their integrity with respect to transepithelial electrical resistance, mannitol and PEG-4000 transport, and cyclosporin efflux. Papp were determined for 20 compounds exhibiting large differences in chemical structure, molecular weight, transport mechanisms, and percentage of absorption in humans. RESULTS: The TC-7 clone exhibits morphological characteristics similar to those of the parental Caco-2 cell line, concerning apical brush border, microvilli, tight junctions and polarisation of the cell line. The TC-7 clone however appeared more homogenous in terms of cell size. Both cell lines achieved a similar monolayer integrity towards mannitol and PEG-4000. Monolayer integrity was achieved earlier for the TC-7 clone, mainly due to its shorter doubling time, i.e. 26 versus 30 hours for parental Caco-2 cells. When using cyclosporin A as a P-glycoprotein substrate, active efflux was lower in the TC-7 clone than in the parental Caco-2 cells. The Papp and mechanisms of transport (paracellular or transcellular routes, passive diffusion and active transport) were determined for 20 drugs. A relationship was established between the in vivo oral absorption in humans and Papp values, allowing to determine a threshold value for Papp of 2 10(-6) cm/sec, above for which a 100% oral absorption could be expected in humans. Both correlation curves obtained with the two cell types, were almost completely superimposable. These studies also confirmed that the dipeptide transporter is underexpressed in both cell lines. CONCLUSIONS: On the basis of morphological parameters, biochemical activity and drug transport characteristics, the TC-7 clone appeared to be a valuable alternative to the use of parental Caco-2 cells for drug absorption studies.
The aim of this study is to evaluate the effects of whole blood filtration after a storage time of 20-24 hours at laboratory temperature using the in line filter Leucoflex LST1. The study concerns 49 blood donations in which we studied leukocyte depletion, proteins (IgG, IgA, IgM, haptoglobin, C3, C4), coagulation factors (fibrinogen, factors XII, XI, IX, VIII, V, proteins S and C, plasminogen, tPA, D-Dimers, PDF) at day 1, the parameters of conservation (ATP, 2-3 DPG, extra cellular potassium, haemolysis, pH) of red blood cell concentrates (RCCs) and bacteriological sterility at day 1 and 42. Despite a correct leukocyte depletion (mean depletion of 3.96 log), a 10 fold higher mean level of residual leukocytes/unit than with buffy coat poor RCC filtration (0.514.10(6) vs 0.051.10(6)) is observed. Moreover a lot of concentrates are not in accordance with French regulations (7/42 with more than 1.10(6) leukocytes/unit). The variation of the rates of IgG, IgA, IgM, haptoglobin, C4 and protein C is not significant. For the others there is a slight decrease with a mean level remaining in a physiological range. No sign of activation is noted. The sterility assays remain negative and the RCC conservation is not altered. In conclusion, even if the quality of the leukocyte depletion is not satisfactory in our study and has to be stated more precisely by multicenter studies, the whole blood filtration does not alter the quality of the derived components and allows us obtain RCC in a bigger volume and containing more haemoglobin than with the classical procedure after removing the buffy-coat [10].
Cytochrome P450 chemical inhibitors are widely used to define the role of individual cytochrome P450 isozyme(s) in a metabolism process. In this study, cytochrome P450 isoform-dependent reactions were investigated on our human liver microsomes bank (n = 34) and characterized for both KM and VMAX values (n > or = 3). These metabolic reactions were: 7-ethoxyresorufin O-deethylation (CYP1A1), phenacetin O-deethylation (CYP1A2), coumarin 7-hydroxylation (CYP2A6), tolbutamide 4-methylhydroxylation (CYP2C9), dextromethorphan O-demethylation (CYP2D6), aniline 4-hydroxylation (CYP2E1) and nifedipine dehydrogenation (CYP3A4). Literature data-based specific inhibitors were selected and characterized for both their inhibitory constant (Ki) and the inhibition-type toward their specific substrate. Results were as follows: alpha-naphthoflavone (CYP1A1; mixed-type interaction with a Ki = 0.01 microM), furafylline (CYP1A2; competitive-type interaction with a Ki = 3 microM when microsomes were incubated with both furafylline and phenacetin; noncompetitive-type interaction with a Ki = 0.6 microM when microsomes were preincubated with furafylline and NADPH), pilocarpine (CYP2A6; competitive-type interaction with a Ki = 4 microM), sulfaphenazole (CYP2C9; competitive-type interaction with a Ki = 0.3 microM), quinidine (CYP2D6; competitive-type interaction with a Ki = 0.4 microM, diallyldisulfide (CYP2E1; noncompetitive-type interaction with a Ki = 150 microM on an aniline concentration range of 10-60 microM; competitive-type interaction with a Ki = 100 microM on an aniline concentration range of 80-2000 microM) and ketoconazole (CYP3A4; mixed-type interaction with a Ki = 0.015 microM). Once the inhibitors' potency was determined, the selective effects of these inhibitors were evaluated after incubation of human hepatic microsomes with isoform-selective substrates in the presence of the different chemical inhibitors. Up to 10 times the Ki value toward the isoform-selective probe, pilocarpine, sulfaphenazole, quinidine and ketoconazole exhibited potent inhibitory and specific effects. alpha-Naphthoflavone and furafylline both inhibited phenacetin and 7-ethoxyresorufin O-deethylation processes, a consequence of the absence of CYP1A1 in noninduced human liver. Diallyldisulfide exhibited broad and nonspecific inhibitory effects. When used in their "window of selectivity," ie., up to 10-fold the Ki value, most chemical inhibitors powerfully and specifically inhibited cytochrome P450 isoform-specific reactions when analyzed at their KM values.
The gastrointestinal tract remains the most popular and acceptable route of administration for drugs. It offers the great advantage of convenience and many compounds are well absorbed and thereby provide acceptable plasma concentration-time profiles. Currently there is considerable interest from the pharmaceutical industry in development of cell culture systems that would mimic the intestinal mucosa in order to evaluate strategies for investigating and/or enhancing drug absorption. The intestinal epithelial cells of primary interest, from the standpoint of drug absorption and metabolism, are the villus cells, which are fully differentiated cells. An in vitro cell culture system consisting of a monolayer of viable, polarized and fully differentiated villus cells, similar to that found in the small intestine, would be a valuable tool in the study of drug and nutrient transport and metabolism. The Caco-2 cell line, which exhibits a well-differentiated brush border on the apical surface and tight junctions, and expresses typical small-intestinal microvillus hydrolases and nutrient transporters, has proven to be the most popular in vitro model (a) to rapidly assess the cellular permeability of potential drug candidates, (b) to elucidate pathways of drug transport (e.g., passive versus carrier mediated), (c) to assess formulation strategies designed to enhance membrane permeability, (d) to determine the optimal physicochemical characteristics for passive diffusion of drugs, and (e) to assess potential toxic effects of drug candidates or formulation components on this biological barrier. Since differentiated Caco-2 cells express various cytochrome P450 isoforms and phase II enzymes such as UDP-glucuronosyltransferases, sulfotransferases and glutathione-S-transferases, this model could also allow the study of presystemic drug metabolism.
The prevalence of serologic markers for hepatitis A was investigated in 936 French male military recruits from October 1992 to June 1993. Data were collected in order to assess the evolution of seroprevalence level according to the decline observed for several years and to appreciate the importance of potential risk factors. The overall prevalence of antibody against hepatitis A virus was 16.3%. The prevalence was higher among those with high number of siblings and whose Father's occupation falls into low professional class categories. Multivariate analysis found that high level of seroprevalence was also associated with tap water consumption (odd ratio (OR) = 1.56; p < 0.04), overseas travels (OR = 2.26; p < 0.001) and was higher for recruits reporting an history of clinical jaundice (OR = 2.27; p < 0.01). Together with more anticipated factors, tap water consumption may be of importance in France and this study points out the potential part taken by chlorinated water.
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The physiological, biochemical, morphological and rheological characteristics of RBC resuspended in PAGGS-Sorbitol were studied over, and beyond, 49 days. The levels of P50 and 2,3-DPG, as well as the concentration of ATP, stayed above acceptable values for longer periods than those described with other Optional Additive Solutions (OAS). Spontaneous hemolysis was kept below 1%, cell morphology, viscosity, deformability and other factors such as pH, the ratio of glucose consumption to lactate formation, the total adenylates and energetic charge indicate that good properties should be seen after 49 days' storage. In vivo survival studies using a double isotope technique showed that at least 75% of those RBC stored in this OAS for 49 days were still in circulation 24 hours after injection. Therefore RBC stored in PAGGS-S can be kept for 49 days with excellent in vivo survival, as well as maintaining DPG levels for up to 3 weeks. Furthermore, a clinical trial in routine conditions for use has been carried out with surgical and medical patients as well as with hemodialysed patients. The results obtained are those that could at least be expected of normal stored blood. As regards RBC/PAGGS-Sorbitol, it would be reasonable to propose a storage period of 49 days in the liquid state.
The glucuronidation of the AT1 nonpeptide angiotensin II receptor antagonist, SR 47436 (BMS 186295), was investigated in hepatic microsomes prepared from various species, i.e., Sprague-Dawley rat, Cynomolgus monkey and Caucasian humans. The drug was found to undergo N-glucuronidation on the tetrazole moiety as confirmed by its hydrolysis by beta-glucuronidase, its associated radioactivity when UDP-[U-14C]glucuronic acid was used as substrate and by different techniques such as high-performance liquid chromatography-mass spectrometry and nuclear magnetic resonance. Glucuronide formation was optimal at pH 5.0 along with a "0.2 mg of Brij 58 per mg of protein" ratio, regardless of the investigated species. Cynomolgus monkey microsomes glucuronidated SR 47436 (BMS 186295) to the greatest extent, with a relative catalytic efficiency 11.0- and 2.6-fold higher than that observed in rat and human, respectively. SR 47436 (BMS 186295) glucuronidation followed Michaelis-Menten kinetics. Bilirubin:UDP-glucuronosyltransferase isoform was not involved, inasmuch as bilirubin did not affect its glucuronidation, 7,7,7-triphenylheptanoic acid was a noncompetitive inhibitor and glucuronidation was only decreased 2-fold in Gunn rats. SR 47436 (BMS 186295) glucuronidation was enhanced markedly after treatment of rats with dexamethasone (Vmax/Km = 71.5 vs. 2.6 in untreated animals). Among the drugs used which undergo phenolic, carboxylic acid, alcohol or tertiary amine glucuronidation, only monodigitoxigenin-monodigitoxoside, flurbiprofen, naproxen, testosterone and estrone inhibited SR 47436 (BMS 186295) glucoronidation in a noncompetitive manner. These data suggest that SR 47436 (BMS 186295) was glucuronidated by a highly dexamethasone-inducible UDP-glucuronosyltransferase isoform(s), different from that involved in the glucuronidation of monodigitoxigenin-monodigitoxoside.
The transport of a new bisphosphonate, Tiludronate, was investigated on the human adenocarcinoma cell line, CACO-2. Experiments were performed 7-16 days after cells achieved confluence, conditions under which they form well-differentiated monolayers joined by tight junctions. Tiludronate transport rate across CACO-2 monolayers was independent of the temperature (4 degrees versus 37 degrees), of the polarity of the cell membrane (apical-to-basolateral versus basolateral-to-apical), and of the presence of metabolic poisons (sodium azide). Its transport was enhanced by either the presence of EGTA in the incubation buffer, i.e. when extracellular Ca2+ concentration was reduced, or by the pretreatment of monolayers with EGTA, i.e. when the intercellular spaces and the tight junctions were widened. Based on these different observations, we could suggest that Tiludronate mainly used the paracellular pathway to cross the intestinal epithelium. An increase in the Tiludronate permeability coefficient was also observed following treatment of cells with high Tiludronate concentrations, as a consequence of the direct effect of this compound on the extracellular Ca2+ ions. Hence, for high drug concentrations, i.e. 20 mM, we observed a decrease in free extracellular Ca2+ concentration, an increase in the transepithelial electrical resistance and an increase in the transport of [14C]polyethyleneglycol ([14C]PEG400), a probe for the paracellular pathway. The results indicate that Tiludronate is transported across CACO-2 monolayers by the paracellular route. Moreover, it can affect its own transport by its concentration-dependent effect on tight junction widening.
The metabolism of 2-n-propyl-2-pentenoate (delta 2-VPA) was evaluated in human hepatic microsomal fractions. Two biotransformation pathways have been particularly investigated. In the presence of the cytochrome P-450 co-factor, NADPH, the main metabolites recovered were delta 3-VPA, delta 2,4-VPA and VPA. The glucuronidation of delta 2-VPA was also studied on various hepatic microsomal fractions using Brij 35 as activator and UDP-glucuronic acid as co-factor. A large interindividual variability occurred in this metabolic pathway. Km and Vmax were 0.85 mmol/l and 1.75 nmol.min-1.mg-1, respectively, for delta 2-VPA and 1.11 mmol/l and 5.71 nmol.min-1.mg-1 for VPA, respectively. The good correlation (r = 0.82; p less than 0.001) observed between the glucuronidation of VPA and delta 2-VPA as well as the mutual inhibition of each other's glucuronidation strongly suggests that (a) common single UDP-glucuronosyltransferase isoenzyme(s) was (were) involved in this glucuronidation step. The glucuronidation of specific substrates for various UDP-glucuronosyltransferase isoenzymes showed a good relationship between the glucuronidations of delta 2-VPA and morphine, a substrate for UDP-glucuronosyltransferase-2B. Moreover, morphine competitively inhibits delta 2-VPA glucuronidation. It seems the same isoenzyme or, at least, (a) very closely related isoenzyme(s) belonging to UDP-glucuronosyltransferase-2 isoenzyme, is involved in delta 2-VPA glucuronidation.
Two cytochrome P450 isozymes, FA and FI, were isolated and characterized from liver microsomes of phenobarbital-induced baboons (Papio papio). The cytochrome FA possesses the same N-terminal amino acid sequence as P450 MK2 from crab-eating monkeys (Macaca irus) and closely resembles the human P450 3A isozymes. This cytochrome was able to oxidize nifedipine and hydroxylate testosterone at the 6 beta position. The second baboon cytochrome (FI) is closely related to the P450 2A subfamily and has the same N-terminal sequence as human P450 2A7. Like human P450 2A forms, it is highly active as a coumarin 7-hydroxylase. Antibodies against P450 FA and FI cross-react with two human liver proteins of 51 kDa and 49 kDa, respectively. The concentration of the first protein in the human samples, was five-times greater than the second. However, the latter showed marked interindividual variation. In primary cultures of human hepatocytes, rifampicin is a strong inducer of the 51-kDa protein and a moderate inducer of the 49-kDa protein, while phenobarbital has the opposite effect on the two proteins.
Hepatitis A antibodies (anti-HAV) were surveyed in 1000 French recruits during 1990. The prevalence of anti-HAV in this group was 21.35%. Compared to a 1985 survey a 9% fall in the anti-HAV prevalence rate was observed. Living in a coastal area, low educational level, stay overseas were the main risk factors, as already noted in 1985.
The expression and inducibility of cytochrome P450IA1 isozyme was investigated in the human carcinoma cell line Caco-2 cultured between days 7 and 35 in the absence or the presence of various enzyme inducers such as 3-methylcholanthrene, beta-naphthoflavone (beta NF), dioxin, isosafrole, rifampycin, dexamethasone or phenobarbital. 7-Ethoxyresorufin O-deethylase activity (EROD) was maximal at day 25 when the differentiation of Caco-2 cells, characterized by the level of the brush border associated enzymes such as sucrase isomaltase and alkaline phosphatase, was higher. The inducibility of this enzyme activity was found to be maximal when cells were treated between days 7 and 10. After a 3-day treatment of Caco-2 cells with 50 microM beta NF, EROD achieved 36.6 +/- 14.6 pmol/min/mg compared to 2.5 +/- 1.1 pmol/min/mg in untreated cells. This enzyme activity appeared to be supported only by P450IA1 isozyme because: 1) EROD was quantitatively inhibited by alpha-naphthoflavone, a P450IA1-specific inhibitor; otherwise, phenacetin O-deethylation was completely abolished in the presence of alpha-naphthoflavone and not by furafylline, a P450IA2-specific inhibitor; 2) EROD was induced after treatment with 3-methylcholanthrene, beta NF and dioxin, which are P450IA1 inducers, but not by isosafrole, a P450IA2-specific inducer; 3) cytochrome P450IA1 apoprotein could be immunodetected by antibodies directed against rabbit cytochrome P450-LM6, orthologous to P450IA1, in polycyclic hydrocarbon-treated cells; 4) under the latter conditions, P450IA1 mRNA accumulation was specifically detected, but not P450IA2 mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)
The transfusion-sheet is an important document which appears first in the French legislation on May 1985 the 17. Its aim is to keep a close watch over transfusion of red blood cells, platelet concentrates and fresh frozen plasma and also immunohematologic evolution of data for every patient. In spite of its importance for everyone in charge of transfusion practice (physicians, biologists, transfusion center,...), a lot of problems may be observed in the strict and complete updating of the document. Informatisation of all the steps of the medical and biological practice appears of crucial interest to perfect its use.